Molecular Determinants of Confined Migration

限制迁移的分子决定因素

• 批准号: 10556661
• 负责人: Cynthia A. Reinhart-King
• 金额: $ 5.8万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10556661
• 关键词: 3-Dimensional; ATP Synthesis Pathway; Actins; Address; Adhesions; Adhesives; Affect; Anatomy; Architecture; Automobile Driving; Behavior; Biological Assay; Biosensor; Cell Adhesion; Cell Energetics; Cell-Matrix Junction; Cells; Cellular Metabolic Process; Chemicals; Collagen; Complex; Confined Spaces; Coupled; Cytoskeleton; Data; Decision Making; Development; Disease; Engineering; Environment; Equilibrium; Event; Extracellular Matrix; Fluorescence Resonance Energy Transfer; Focal Adhesions; Foundations; Gel; Heterogeneity; Image; Immune response; In Vitro; Individual; Intervention; Matrix Metalloproteinases; Measurement; Mechanics; Mediating; Metabolic; Metabolic Pathway; Metabolism; Metastatic to; Microfabrication; Modeling; Molds; Molecular; Monitor; Motion; Movement; Natural Products; Neoplasm Metastasis; Nutrient; Organ; Pattern; Pharmacology; Physiological; Play; Population; Process; Publishing; Research; Role; Shapes; Site; Structure; System; Talin; Techniques; Tissues; Vinculin; Work; base; cell motility; cellular imaging; chemical property; cost; design and construction; in vitro Model; in vivo; interstitial; mechanical properties; migration; mutant; new therapeutic target; novel; optogenetics; rho GTP-Binding Proteins; therapeutic target; transmission process; tumor

Project Summary: In numerous processes including development and metastasis, cells can move in microtracks within the 3D microenvironment. These microtracks are formed by cells themselves through the use of matrix metalloproteinases that degrade matrix, or microtracks can exist as a product of the natural architecture of organs. While microtrack migration occurs in vivo, little is known about the specific mechanisms that cells employ to move in microtracks. We have developed a unique platform using microfabrication to recreate these microtracks in vitro by micromolding collagen. Microtracks can be made in various sizes, and they can be patterned into multiple different shapes including tapered channels and bifurcated channels. Our microfabricated microtracks are structurally indistinguishable from tracks found in vitro and in vivo. Moreover, they offer a distinct advantage over other PDMS-based platforms because the collagen is amenable to cell adhesion on all 4 walls of the track, the fibrous walls of the microtrack can be deformed by cells, and the tracks more closely mimic the mechanical and chemical properties found in vivo. Importantly, our work to-date has shown that the mechanisms driving movement in microtracks are not the same as those mediating cell migration on 2D substrates or in unmolded collagen. Here, we propose to build upon two of our major prior findings, which are that: 1. Vinculin is required for microtrack movement, 2. Cellular confinement alters migration and correlates with cell metabolism. Using this novel microtrack platform in concert with engineered probes to monitor adhesion and cellular energy, optogenetic probes to alter cell contractility and cellular protrusions, and novel force measurement techniques, we will investigate the molecular mechanisms driving cell migration and decision-making during migration in microtracks with a focus on adhesion dynamics and cellular energetics. In Aim 1, we investigate the role of focal adhesion dynamics and tension, focusing on vinculin-talin-actin interactions based on our preliminary showing vinculin mediates unidirectional motion. We will investigate the linkage between vinculin, talin and actin, and we will probe the force transmission occurring at the sites of cell-matrix adhesion. In Aim 2, we will investigate how cellular energetics and the availability of nutrients affects migration and migration decisions in confined spaces. Based on our prior work indicating that the extracellular matrix structure alters ATP utilization, we hypothesize that increased confinement will increase the energetic needs of the cell. In Aim 3, we will investigate the molecular and mechanical mechanisms governing cell migration decisions. Constructs designed to disrupt force transmission between the cell and the matrix and pharmacological interventions will be used to assess the effects of cell contractility and cell stiffness on cellular energy utilization, adhesion, and migration direction decisions in microtracks. Our understanding of metabolism is rapidly developing, and as such, therapeutics targeting metabolic pathways are emerging. Connecting migration behaviors to metabolism offers a potential new point of intervention in disease.

项目概述：在包括发育和转移在内的许多过程中，细胞可以进入 3D微环境中的微轨道。这些微轨道是由细胞自身通过使用 降解基质的基质金属蛋白酶，或微轨道可以作为天然结构的产物存在， 的器官。虽然微轨道迁移发生在体内，但对细胞迁移的具体机制知之甚少。 在微轨道上移动。我们开发了一个独特的平台，使用微加工来重现这些 通过微成型胶原蛋白在体外进行微跟踪。微轨道可以制成各种尺寸，并且它们可以 图案化成包括锥形通道和分叉通道的多种不同形状。我们的微加工 微轨道在结构上与体外和体内发现的轨道无法区分。此外，它们还提供了一种独特的 优于其他基于PDMS的平台，因为胶原蛋白易于细胞粘附在所有4个壁上 微道的纤维壁可以被细胞变形，并且微道更接近地模仿微道的纤维壁。 在体内发现的机械和化学性质。重要的是，我们迄今为止的工作表明， 微轨道中的驱动运动与介导2D基底上的细胞迁移或微轨道中的细胞迁移的那些不同。 未成型的胶原蛋白。在这里，我们建议建立在我们的两个主要的先前发现，这是：1。纽蛋白 是微轨道运动所必需的，2.细胞限制改变迁移并与细胞代谢相关。 使用这种新型的微轨道平台与工程探针配合监测粘附和细胞能量， 改变细胞收缩性和细胞突起的光遗传学探针，以及新的力测量技术， 我们将研究驱动细胞迁移和迁移过程中决策的分子机制， microtracks专注于粘附动力学和细胞能量学。在目标1中，我们研究了焦点的作用。 粘附动力学和张力，侧重于纽蛋白-talin-肌动蛋白的相互作用，根据我们的初步显示 黏着斑蛋白介导单向运动。我们将研究黏着斑蛋白、talin和肌动蛋白之间的联系， 将探测在细胞-基质粘附位点发生的力传递。在目标2中，我们将研究如何 细胞能量学和营养物质的可获得性影响着有限空间中的迁移和迁移决定。 基于我们先前的工作表明细胞外基质结构改变ATP利用，我们假设 增加的限制将增加细胞的能量需求。在目标3中，我们将研究 控制细胞迁移决定的分子和机械机制。设计用来破坏原力的结构 将使用细胞和基质之间的传输和药理学干预来评估效果 细胞收缩性和细胞硬度对细胞能量利用、粘附和迁移方向的决定， 微轨我们对新陈代谢的理解正在迅速发展，因此， 代谢途径正在出现。将迁移行为与新陈代谢联系起来提供了一个潜在的新观点， 干预疾病。

项目成果

Matrix-driven changes in metabolism support cytoskeletal activity to promote cell migration.

基质驱动的代谢变化支持细胞骨架活性，促进细胞迁移。

• DOI: 10.1016/j.bpj.2021.02.044
• 发表时间: 2021
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Wu,Yusheng;Zanotelli,MatthewR;Zhang,Jian;Reinhart-King,CynthiaA
• 通讯作者: Reinhart-King,CynthiaA

Cellular mechanosignaling for sensing and transducing matrix rigidity.

• DOI: 10.1016/j.ceb.2023.102208
• 发表时间: 2023-07
• 期刊: Current opinion in cell biology
• 影响因子: 7.5
• 作者: Katherine M. Young;Cynthia A. Reinhart-King